Throughout this application, various references are referred to within parentheses. Disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains. Full bibliographic citations for these references may be found at the end of this application, preceding the sequence listing and claims.
Vaccinia topoisomerase binds duplex DNA and forms a covalent DNA-(3xe2x80x2-phosphotyrosyl)-protein adduct at the sequence 5xe2x80x2-CCCTT1. The enzyme reacts readily with a 36-mer CCCTT strand (DNA-p-RNA) composed of DNA 5xe2x80x2 and RNA 3xe2x80x2 of the scissile bond. However, a 36-mer composed of RNA 5xe2x80x2 and DNA 3xe2x80x2 of the scissile phosphate (RNA-p-DNA) is a poor substrate for covalent adduct formation. Vaccinia topoisomerase efficiently transfers covalently held CCCTT-containing DNA to 5xe2x80x2xe2x80x94OH terminated RNA acceptors; the topoisomerase can therefore be used to tag the 5xe2x80x2 end of RNA in vitro.
Religation of the covalently bound CCCTT-containing DNA strand to a 5xe2x80x2xe2x80x94OH terminated DNA acceptor is efficient and rapid (krel greater than 0.5 secxe2x88x921), provided that the acceptor DNA is capable of base-pairing to the noncleaved DNA strand of the topoisomerase-DNA donor complex. The rate of strand transfer to DNA is not detectably affected by base mismatches at the 5xe2x80x2 nucleotide of the acceptor strand. Nucleotide deletions and insertions at the 5xe2x80x2 end of the acceptor slow the rate of religation; the observed hierarchy of reaction rates is: +1 insertion  greater than xe2x88x921 deletion  greater than +2 insertion  greater than  greater than xe2x88x922 deletion. These findings underscore the importance of a properly positioned 5xe2x80x2 OH terminus in transesterification reaction chemistry, but also raise the possibility that topoisomerase may generate mutations by sealing DNA molecules with mispaired or unpaired ends.
Vaccinia topoisomerase, a 314-amino acid eukaryotic type I enzyme, binds and cleaves duplex DNA at a specific target sequence 5xe2x80x2-(T/C)CCTT1 (1-3). Cleavage is a transesterification reaction in which the Tp1N phosphodiester is attacked by Tyr-274 of the enzyme, resulting in the formation of a DNA-(3xe2x80x2-phosphotyrosyl) protein adduct (4). The covalently bound topoisomerase catalyzes a variety of DNA strand transfer reactions. It can religate the CCCTT-containing strand across the same bond originally cleaved (as occurs during the relaxation of supercoiled DNA) or it can ligate the strand to a heterologous acceptor DNA 5xe2x80x2 end, thereby creating a recombinant molecule (5-7).
Duplex DNA substrates containing a single CCCTT target site have been used to dissect the cleavage and strand transfer steps. A cleavage-religation equilibrium is established when topoisomerase transesterifies to DNA ligands containing xe2x89xa718-bp of duplex DNA 3xe2x80x2 of the cleavage site (8-11). The reaction is in equilibrium because the 5xe2x80x2xe2x80x94OH terminated distal segment of the scissile strand remains poised near the active site by virtue of the fact that it is stably base-paired with the nonscissile strand. About 20% of the CCCTT-containing strand is covalently bound at equilibrium (11). xe2x80x9cSuicidexe2x80x9d cleavage occurs when the CCCTT-containing substrate contains no more than fifteen base pairs 3xe2x80x2 of the scissile bond, because the short leaving strand dissociates from the protein-DNA complex. In enzyme excess,  greater than 90% of the suicide substrate is cleaved (11).
The suicide intermediate can transfer the incised CCCTT strand to a DNA acceptor. Intramolecular strand transfer occurs when the 5xe2x80x2 xe2x80x94OH end of the noncleaved strand of the suicide intermediate attacks the 3xe2x80x2 phosphotyrosyl bond and expels Tyr-274 as the leaving group. This results in formation of a hairpin DNA loop (5). Intermolecular religation occurs when the suicide intermediate is provided with an exogenous 5xe2x80x2xe2x80x94OH terminated acceptor strand, the sequence of which is complementary to the single strand tail of the noncleaved strand in the immediate vicinity of the scissile phosphate (5). In the absence of an acceptor strand, the topoisomerase can transfer the CCCTT strand to water, releasing a 3xe2x80x2-phosphate-terminated hydrolysis product, or to glycerol, releasing a 3xe2x80x2-phosphoglycerol derivative (12). Although the hydrolysis and glycerololysis reactions are much slower than religation to a DNA acceptor strand, the extent of strand transfer to non-DNA nucleophiles can be as high as 15-40%.
The specificity of vaccinia topoisomerase in DNA cleavage and its versatility in strand transfer have inspired topoisomerase-based strategies for polynucleotide synthesis in which DNA oligonucleotides containing CCCTT cleavage sites serve as activated linkers for the joining of other DNA molecules with compatible termini (13). The present study examines the ability of the vaccinia topoisomerase to cleave and rejoin RNA-containing polynucleotides. It was shown previously that the enzyme did not bind covalently to CCCTT-containing molecules in which either the scissile strand or the complementary strand was composed entirely of RNA (9). To further explore the pentose sugar specificity of the enzyme, we have prepared synthetic CCCTT-containing substrates in which the scissile strand is composed of DNA-and RNA-containing halves. In this way, we show that the enzyme is indifferent to RNA downstream of the scissile phosphate, but is does not form the covalent complex when the region 5xe2x80x2 of the scissile phosphate is in RNA form. Also assessed is the contribution of base-pairing by the 5xe2x80x2 end of the acceptor strand to the rate of the DNA strand transfer reaction.
The present invention provides a method of covalently joining a DNA strand to an RNA strand comprising (a) forming a topoisomerase-DNA intermediate by incubating a DNA cleavage substrate comprising a topoisomerase cleavage site with a topoisomerase specific for that site, wherein the topoisomerase-DNA intermediate has one or more 5xe2x80x2 single-strand tails; and (b) adding to the topoisomerase-DNA intermediate an acceptor RNA strand complementary to the 5xe2x80x2 single-strand tail under conditions permitting a ligation of the 5xe2x80x2 single-strand tail of the topoisomerase-DNA intermediate to the RNA acceptor strand and dissociation of the topoisomerase, thereby covalently joining the DNA strand to the RNA strand. The DNA cleavage substrate may be created by hybridizing a DNA strand having a topoisomerase cleavage site to one or more complementary DNA strands, thereby forming a DNA cleavage substrate having a topoisomerase cleavage site and a oligonucleotide leaving group located 3xe2x80x2 of a scissile bond or may be a plasmid vector comprising a topoisomerase cleavage site.
The present invention also provides a covalent topoisomerase-DNA intermediate having a 5xe2x80x2 single-strand tail.
Another aspect of the present invention provides a DNA-RNA molecule covalently joined by topoisomerase catalysis.
The present invention provides a covalently joined DNA-RNA molecule having a labeled 5xe2x80x2 end.
The present invention further provides a method of tagging a 5xe2x80x2 end of an RNA molecule comprising: (a) forming a topoisomerase-DNA intermediate by incubating a DNA cleavage substrate comprising a topoisomerase cleavage site with a topoisomerase specific for that site, wherein the topoisomerase-DNA intermediate has one or more 5xe2x80x2 single-strand tails; and (b) adding to the topoisomerase-DNA intermediate a 5xe2x80x2-hydroxyl terminated RNA molecule complementary to the 5xe2x80x2 single-strand tail under conditions permitting a ligation of the covalently bound DNA strand of the topoisomerase-DNA intermediate to the RNA molecule and dissociation of the topoisomerase, thereby forming a 5xe2x80x2 end tagged DNA-RNA ligation product. The DNA cleavage substrate can be created, for example, by hybridizing a DNA strand having a topoisomerase cleavage site to a complementary DNA strand, thereby forming a DNA cleavage substrate having a topoisomerase cleavage site and a oligonucleotide leaving group located 3xe2x80x2 of a scissile bond.
Another aspect of the present invention provides a 5xe2x80x2 end tagged RNA molecule.
In another aspect the present invention also provides a DNA-RNA molecule which has been joined in vitro by the use of a topoisomerase.
The present invention further provides a method of tagging a 5xe2x80x2 end of a capped messenger RNA comprising:
a) isolating mRNA from cells or a tissue; b) removing an RNA cap structure from the isolated mRNA, resulting in a de-capped RNA; c) dephosphorylating the de-capped RNA, thereby forming a de-capped and dephosphorylated RNA;
d) constructing a DNA cleavage substrate for topoisomerase having a topoisomerase cleavage site and a complementary strand, the complementary strand having a mixed or random base composition downstream of the topoisomerase cleavage site, the DNA cleavage substrate being designated as a DNA-(N) substrate; e) cleaving the DNA-(N) substrate with a topoisomerase, thereby forming a covalent topoisomerase-DNA-(N)M complex containing a 5xe2x80x2 tail of mixed or random base composition on a noncleaved strand; and f) incubating the cleaved covalent topoisomerase-DNA-(N)M complex with the de-capped and dephosphorylated RNA formed in step (c) to form a 5xe2x80x2 DNA-tagged DNA-RNA ligation product.
As used herein the number of bases (N) of the DNA cleavage substrate, designated supra as a DNA-(N) substrate, may be from one to four bases long.
The present invention also provides a method of isolating and cloning a capped mRNA after subtraction of non-capped RNA comprising: a) isolating mRNA from cells or a tissue;
b) dephosphorylating the mRNA; c) incubating a cleaved topoisomerase-BioDNA-(N) complex with the dephosphorylated mRNA to form a 5xe2x80x2 BioDNA-tagged DNA-RNA ligation product;
d) removing the 5xe2x80x2 BioDNA-tagged DNA-RNA ligation product and any unreacted cleaved topoisomerase-BioDNA-(N) complex by adsorption to streptavidin and recovering any nonadsorbed material, said material being enriched for RNA having a capped 5xe2x80x2 end and being resistant to dephosphorylation in step (b), thereby being unable to react with the cleaved topoisomerase-BioDNA-(N) complex; e) removing of the 5xe2x80x2 end cap from the enriched RNA recovered from the nonadsorbed material in step (d); f) dephosphorylating the de-capped RNA, thereby forming a de-capped and dephosphorylated RNA; g) incubating a cleaved topoisomerase-BioDNA-(N) complex with the de-capped and dephosphorylated RNA to form a 5xe2x80x2 BioDNA-tagged DNA-RNA ligation product; h) affinity purifying the 5xe2x80x2 DNA-tagged DNA-RNA ligation product; and i) PCR amplification of the decapped and dephosphorylated RNA of the DNA-RNA ligation product using a sense primer corresponding to a scissile strand of the topoisomerase cleavage substrate 5xe2x80x2 of the site of cleavage and an antisense primer, said antisense primer being complementary to either a 3xe2x80x2 poly(A) tail or to an internal RNA sequence.
The present invention also provides a method of obtaining full-length gene sequences comprising attaching a DNA tag to an isolated mRNA sequence and using the DNA-tagged mRNA as a template for DNA synthesis. DNA may be further inserted into an expression vector and used to express recombinant protein.